Purpose: :
While the presence of a few small ‘hard’ drusenis a normal part of aging, the deposition of large, diffusedrusen in the macula is indicative of early age-related maculardegeneration (AMD). Although the composition of drusen has beenwidely examined, flat mount analysis of their influence on theoverlying RPE monolayer has been limited. In this study we analyzedmorphometric and autofluorescence features of human RPE flatmounts on and around drusen by confocal microscopy.

Methods: :
Human eyes obtained from 46-77 year old donors, with and withouta history of AMD, were supplied by the North Carolina Eye Bankand fixed in 4% paraformaldehyde. Six mm diameter punches weretaken from the macula, and central and equatorial retina. Thesclera and retina were removed, and the punch was placed RPEside down. The choroid and Bruch’s membrane were removedto expose the basal surface of the RPE. Some RPE flat mountswere stained with Hoeschst. RPE images were acquired using aLeica SP5 confocal microscope. Morphometric analysis and spectralmeasurements were performed with the Leica Application Suite,ImageJ and the Duke Ophthalmic Cell Analysis Program.

Results: :
Typical morphology of the RPE included tightly joined hexagonalcells containing single nuclei and autofluorescent lipofuscingranules. These aggregates exhibited characteristic emissionspectra with a peak around 530 nm under excitation by a 405nm laser. In contrast, atypical changes found in associationwith drusen included cell enlargement, multi-nucleate cells,hyper- and hypo-pigmentation and distortions of the RPE sheet.Interestingly, the autofluorescent spectra of some abnormalcells were blue-shifted with a peak around 500 nm in comparisonto healthy cells, and these resembled the measured spectra ofsub-RPE deposits.

Conclusions: :
RPE flat mounts imaged from the basal side is an informativetechnique for assessing the negative impact of drusen on theRPE monolayer. This approach represents a novel perspectivefor imaging aging- and AMD-related changes to the RPE.